The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence

Abstract

Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro. A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells

“…Typhimurium T3SS-effector proteins that have been previously shown to have the capacity to modulate this signaling pathway. Expression of spvD , which encodes a cysteine protease that has been reported to negatively regulate nuclear transport of NF-κB components [ 32 , 33 ], did not affect the ability of S . Typhi to stimulate NF-κB signaling ( Fig 4B ).…”

Bimodal Expression of theSalmonellaTyphimuriumspvOperon

“…In particular, SpvB has been characterized as an (ADP-ribosyl)transferase, activating host cell actin degradation and thereby mediating cytotoxicity in macrophages and virulence in mice (Mazurkiewicz et al 2008), while SpvC was found to exhibit phosphothreonine lyase activity on host mitogen-activated protein kinases (MAPKs), leading to attenuation of the intestinal inflammatory response, which is thought to be important during systemic infection of S. typhimurium (Otto et al 2000). Recently, the function and crystal structure of the SpvD protein were elucidated and it was found that SpvD acts as a cysteine protease, which downregulates proinflammatory responses by indirectly inhibiting NF-kB regulated promoters and by doing so contributes to the systemic growth of S. typhimurium in mice (Grabe et al 2016;Rolhion et al 2016). In contrast, little is known about the SpvA protein and it is yet unclear if it is involved in Salmonella virulence (Roudier et al 1992;Rotger and Casadesus 1999).…”

How to rewire the host cell: A home improvement guide for intracellular bacteria

“…1 B ). The crystal structure and mutational analysis of SpvD suggest that SpvD is a cysteine protease; however, exactly how this protease function negatively regulates NF-κB signal transduction is still unknown ( Grabe et al, 2016 ). Overall, many intracellular bacterial pathogens secrete effectors that target MAPK and NF-κB signaling pathways to counteract the deleterious output of these host defense pathways and prevent downstream proinflammatory cytokine expression.…”